Glucose and pH are two important indicators of diabetes mellitus. However, their dynamic changes at the same time in brain are still not clear, mainly due to a lack of a single biosensor capable of simultaneous quantification of two species in a live rat brain. In this work, a selective and sensitive ratiometric electrochemical biosensor was developed for simultaneously quantifying glucose and pH using both current and potential outputs in a rat brain of diabetic model. Here, glucose oxidase was first employed as a specific recognition element for both glucose and pH because the active center (FAD) could undergo a 2H+/2e- process. Moreover, an insensitive molecule toward pH and glucose was used as an inner-reference element to provide a built-in correction to improve the accuracy. The ratio between the oxidation peak current density of glucose and that of ABTS gradually increased with increasing concentration of glucose, and showed a good linearity in the range of 0.3-8.2 mM. Meanwhile, the midpotential difference between glucose oxidase and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) positively shifted with pH decreasing, leading to accurate determination of pH in the linear range of 5.67-7.65. Thus, combined with the unique properties of carbon fiber microelectrode, including easy to insert and good biocompatibility, the developed single biosensor was successfully applied to detect pH and glucose at the same time in hippocampus, striatum, and cortex in a live rat brain of diabetic model.